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Journal of Chinese Integrative Medicine ›› 2009, Vol. 7 ›› Issue (9): 860-867.doi: 10.3736/jcim20090912

• Original Experimental Research • Previous Articles     Next Articles

Effects of Naomaitong combined with mobilization of bone marrow mesenchymal stem cells on neuron apoptosis and expressions of Fas, FasL and caspase-3 proteins in rats with cerebral ischemia

Jian-sheng Lia,Jing-xia Liub,Yu-shou Tiana,Wei-hong Renc,Xin-feng Zhanga,Ding-chao Wanga   

  1. a Institute of Geriatrics, Henan College of Traditional Chinese Medicine, Zhengzhou 450008, Henan Province, China
    b School of Traditional Chinese Medicine, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
    c Clinical Laboratory, the First Affiliated Hospital, Henan College of Traditional Chinese Medicine, Zhengzhou 450006, Henan Province, China
  • Received:2009-05-04 Accepted:2009-07-26 Online:2009-09-20 Published:2009-09-15
  • Contact: Jian-sheng Li E-mail:li_js8@163.com

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Objective

To observe the effects of Naomaitong, a compound traditional Chinese herbal medicine, combined with mobilization of bone marrow mesenchymal stem cells (BMSCs) on neuron apoptosis in rats with cerebral ischemia, and to explore the possible mechanism by detecting the expressions of Fas, FasL and caspase-3 proteins.
Methods

Two hundred and two SD rats were divided into sham-operated group, untreated group, recombinant granulocyte colony-stimulating factor (rG-CSF) group, Naomaitong group and Naomaitong plus rG-CSF group (combination group). Focal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion using a nylon thread with some modification. Rats in the rG-CSF group and the untreated group were administered with rG-CSF 10 μg/(kg·d) by subcutaneous injection 3 d before and 2 d after the operation respectively, once a day, and rats in the Naomaitong group and the combination group were intragastrically administered Naomaitong before and after the operation until sacrificed. Two, three, seven and fourteen days after operation, count of CD34-positive cells in peripheral blood and CD34 expression in brain tissue were determined. General neural function score (GNFS) was evaluated. Neuron apoptosis, expressions of Fas, FasL and caspase-3 in rat’s brain were all measured.
Results

Count of CD34-positive cells in peripheral blood and CD34 expression in brain tissue were high in the untreated group, and reached the peak at 3 d and 7 d respectively. CD34 expression in brain tissue was increased in each treated group, especially in the combination group. GNFS was increased at 3 d and 7 d in the untreated group, 7 d and 14 d in the rG-CSF group and the combination group. Expressions of Fas, FasL and caspase-3 were increased 2, 3 and 7 d after operation, while expression of FasL at 2 d in the rG-CSF group, expressions of Fas, FasL and caspase-3 in the combination group were decreased. Expressions of Fas, FasL and caspase-3 at 7 d and 14 d in the combination group were lower than those in the rG-CSF group. Meanwhile, expressions of Fas, FasL and caspase-3 were decreased in each group at 14 d as compared with those at 3 d.
Conclusion

There exists interaction between Naomaitong and BMSC mobilization in the effect of improving nerve function and inhibiting neuron apoptosis in rats after cerebral ischemia. It is implied that Naomaitong combined with BMSC mobilization down-regulates the expressions of Fas and FasL in early phase and then inhibits the apoptosis cascade reaction caused by caspase-3, which causes further inhibition of Fas and FasL expression after cerebral ischemia.

Key words: Cerebral ischemia, Naomaitong, Recombination granulocyte colony-stimulating factor, Bone marrow stem cells, Apoptosis, Rats

Table 1

Count of CD34-positive cells in peripheral blood and expression of CD34 in brain tissues of rats in each group （?x±s）"

Group Time (d) n Count of CD34-positive cells in
peripheral blood (cells/μL)		 n Expression of CD34 in brain tissues (IOD)
Sham-operated 14 8 1.17±0.41 8 —
Untreated 2 7 3.17±0.75* 7 22.04±2.95**
3 7 5.00±1.55*■ 7 25.69±2.44**
7 6 2.33±1.21☆ 6 33.04±2.62**■■☆
14 6 1.33±1.03■☆☆ 6 16.91±2.95**■■☆★★
Naomaitong 2 7 3.33±1.03□□ 7 25.16±1.58△△□□
3 6 5.67±1.03□□■ 6 28.15±1.22△△□□
7 7 2.50±1.05□□☆ 7 34.61±1.20△△□□■■☆☆
14 6 1.67±0.82□□■☆☆ 6 19.88±1.62△△□□■☆☆★★
rG-CSF 2 8 8.83±2.14△▲ 7 43.21±4.41△△▲▲
3 7 13.50±1.87△△▲▲□□■■ 7 45.79±1.76△△▲▲
7 7 5.33±1.21△△▲▲□□■☆☆ 7 52.09±2.86△△▲▲■■☆
14 6 2.00±0.89□□■■☆★ 6 29.73±1.99△△▲▲■■☆☆★★
Combination 2 8 8.50±2.43△△ 7 45.04±2.04△△
3 7 15.83±1.17△△■■ 6 52.47±1.97△△■■
7 7 7.50±1.05△△☆☆ 6 61.73±2.67△△■■☆☆
14 7 3.17±0.75△■☆☆★★ 7 36.16±2.11△△■■☆☆★★

Table 2

General neural function score of rats in each group （?x±s）"

Group Time (d) n Score of general
neural function
Sham-operated 14 9 18.00±0.00
Untreated 2 8 8.14±0.69**
3 8 9.13±0.54**
7 7 10.53±0.76**■■☆
14 7 12.38±0.52**■■☆☆★★
Naomaitong 2 9 9.50±0.54△△
3 9 10.75±0.71△■
7 8 12.29±0.76△△□■■☆☆
14 7 14.03±0.64△△□□■■☆☆★
rG-CSF 2 8 8.71±0.76▲□
3 8 9.59±0.49▲□
7 7 11.86±0.69△□□■■☆☆
14 7 13.38±0.52△△□□■■☆☆★
Combination 2 8 9.48±0.64△△
3 7 10.88±0.84△△■
7 7 13.71±0.76△△■■☆☆
14 8 15.13±0.64△△■■☆☆★★

Table 3

Count of apoptosis cells and expression of caspase-3 in brain tissues of rats in each group （?x±s）"

Group Time (d) n Count of apoptosis cells
(cells/field)		 n Expression of caspase-3(IOD)
Sham-operated 14 8 6.77±1.84 7 17.76±3.91
Untreated 2 7 86.25±5.26** 7 31.64±6.78**
3 7 58.24±13.88** 7 29.51±8.10**
7 6 39.97±1.73**■■ 6 25.49±5.82**
14 6 37.47±2.68**■■ 6 22.68±4.28**■
Naomaitong 2 7 64.33±10.71△△ 7 27.89±2.79
3 7 49.03±9.88□ 7 24.44±7.70▲
7 7 36.62±5.04□□ 6 18.68±4.25△□□
14 6 25.06±7.43△□□■■ 6 16.16±3.62△□□■■
rG-CSF 2 7 67.97±11.08△△▲▲ 7 28.03±4.16
3 7 46.53±3.41▲□□ 6 24.98±4.30
7 7 35.96±4.13□□ 7 22.24±3.02▲▲□
14 6 34.03±6.03□□■ 6 19.27±5.06▲□□■
Combination 2 7 50.46±7.62△△ 6 24.46±3.97△
3 6 35.73±5.41△△ 6 18.02±3.21△△
7 6 29.49±6.04△△ 6 14.69±4.25△△
14 7 23.56±12.37△■ 7 12.80±3.05△△■

Table 4

Expressions of Fas and FasL in brain tissues of rats in each group （?x±s, IOD）"

Group Time (d) n Fas n FasL
Sham-operated 14 7 23.55±5.04 7 24.77±3.34
Untreated 2 7 42.57±6.55** 7 43.78±5.05**
3 7 37.14±7.16** 7 38.05±2.98**
7 6 32.98±5.25**■■ 6 34.55±10.54*■
14 6 28.37±4.24■■☆ 6 26.76±3.00■■☆☆★
Naomaitong 2 7 35.94±5.38 7 38.26±9.74□
3 7 30.48±4.90 7 32.16±7.99□
7 7 25.92±3.37△□■■ 6 25.76±5.34△□■■
14 6 21.31±3.98△□■■☆☆ 6 19.10±3.62△■■☆☆
rG-CSF 2 7 40.63±11.28 7 30.65±3.06△△
3 7 36.83±7.87□□ 6 33.59±3.74□□
7 7 27.82±6.23□☆☆ 7 26.44±7.79□
14 6 22.24±4.60□■☆☆ 6 22.84±9.95□■☆☆
Combination 2 7 30.41±6.67△△ 6 28.75±8.91△△
3 6 24.35±5.58△△ 6 23.46±6.58△△
7 6 18.94±5.05△△■■ 6 16.78±3.21△△■■
14 7 13.78±1.84△△■■☆☆ 7 14.51±3.69△△■■☆
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